Rotationally Actuated Magnetic Bead Trap and Mixer

ABSTRACT

A magnetic bead trap-and-mixer includes a channel having openings at opposing ends, and a rotor adjacent to the channel and comprising a permanent magnet, wherein the rotor is adapted to apply a magnetic field to the channel of sufficient strength to direct the movement of magnetic beads therein. In aspects, the channel is straight and/or has narrowed end. In further aspects, the rotor generates in the channel areas of areas of strong magnetic fields alternating with areas of very weak magnetic fields and the strong magnetic fields extend entirely across the channel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of U.S. Provisional Application No.61/299,587 filed on Jan. 29, 2010, the entirety of which is incorporatedherein by reference.

BACKGROUND

Magnetic beads have become a popular means of performing affinityseparations and bioprocessing reactions. The beads can be pulled fromsuspension by applying a permanent magnet to the side of a vesselcontaining them. Many of the current protocols are not automated andstill require the manual addition of reagents, collection, andresuspension of the beads. Automation usually involves the use of largeelectromagnets, which can be placed at the side of a tube or capillaryto collect the beads and subsequently turned off so to release thebeads. However, the currents typically required preclude their use inbattery powered devices. Added engineering is also typically needed tomake sure the heat generated by the coils does not interfere with thechemistry of the beads. These prior designs also do not provide anymixing of the beads with the solution while they are trapped. Certainprior designs also cause undesired aggregation of magnetic beads and/orfail to release the beads concentrated into a reduced volume as desired.

A need exists for a mechanically simple means of capturing magneticbeads from a flowing stream, providing some degree of mixing with thepassing fluid, and releasing the beads back into the stream whileminimizing aggregation.

BRIEF SUMMARY

In one embodiment, a magnetic bead trap-and-mixer includes a straightchannel having openings at opposing ends, and a rotor adjacent to thechannel and comprising a permanent magnet, wherein the rotor is adaptedto apply a magnetic field to the channel of sufficient strength todirect the movement of magnetic beads therein.

In one embodiment, a magnetic bead trap-and-mixer includes a channelhaving openings at opposing ends and a diameter that is narrower nearthe opposing ends than in a center of the channel, and a rotor adjacentto the channel and comprising a permanent magnet, wherein the rotor isadapted to apply a magnetic field to the channel of sufficient strengthto direct the movement of magnetic beads therein

In another embodiment, a magnetic bead trap-and-mixer includes a channelhaving openings at opposing ends, and a rotor adjacent to the channeland comprising a permanent magnet, wherein the rotor is adapted to applya magnetic field to the channel of sufficient strength to direct themovement of magnetic beads therein, and the rotor generates in thechannel areas of areas of strong magnetic fields alternating with areasof very weak magnetic fields and the strong magnetic fields extendentirely across the channel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary embodiment of a magnetic bead trap-and-mixer.

FIG. 2 shows the “catch and release” mixing of magnetic beads.

FIG. 3 shows the release of magnetic beads.

FIG. 4 shows the magnetic fields resulting from a rotor wherein themagnetic poles are arranged to focus the magnetic fields to a point.

FIG. 5 shows the magnetic fields in an embodiment having magnetsarranged in an alternating configuration.

FIG. 6 shows how a linear magnetic field may be used to move the beadsacross a channel as well as longitudinally upstream or downstream.

FIG. 7 contains images wherein magnetic filings are used to visualizethe magnetic fields of magnets arranged in various configurations.

FIG. 8 shows bead capture results for magnets in various configurations.

DETAILED DESCRIPTION

Definitions

Before describing the present invention in detail, it is to beunderstood that the terminology used in the specification is for thepurpose of describing particular embodiments, and is not necessarilyintended to be limiting. Although many methods, structures and materialssimilar, modified, or equivalent to those described herein can be usedin the practice of the present invention without undue experimentation,the preferred methods, structures and materials are described herein. Indescribing and claiming the present invention, the following terminologywill be used in accordance with the definitions set out below.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” do not preclude plural referents, unless thecontent clearly dictates otherwise.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items.

Description

The apparatus and method described herein aims to concentrate magneticbeads and expose them to one or more fluids with minimal beadaggregation. This is important both for maximizing the efficiency ofdifferent bead surface reactions and for the ability to interrogateindividual beads in analytical equipment downstream from the device. Thebeads may be mixed with a sample to be analyzed or a reagent forprocessing prior to introduction into the trap or the beads may besuspended in a fluid within the trap prior to the addition of a sampleor reagent. In the first case, the beads will be concentrated in thetrap as the higher volume of sample or reagent passes through thechannel. In the second case, the trap would retain the beads in aconcentrated suspension as sample and/or reagents are passed through thechannel. After the processing is complete, the concentrated beads arereleased into downstream analytical equipment including but not limitedto flow cytometers, imaging devices, spectrometers, impedance meters,microarray analyzers, or electrochemical sensors. Alternatively, thereleased beads with any bound cells or molecules can be retained forcell culture or other further processing.

A rotor incorporating one or more permanent magnets rotates adjacent toa channel adapted to contain magnetic beads in a liquid. When therotation results in a magnetic field passing across the channelgenerally in a direction opposite to flow of the liquid, the beads areeffectively trapped and mixed in the liquid. By changing the directionof rotation, the beads can be released from the channel.

In one aspect, the rotor includes a single permanent magnet that wrapsaround the channel, for example with a horse-shoe shape. In otheraspects, one or more magnets are included in the rotor.

The rotor can be placed so that the plane of rotation is parallel to theaxis of the channel (or the plane of the channel if the channel iscurved or arced), or it may be tilted, so that magnets are closest tothe channel in a region where trapping is desired and move away from thechannel where release is desired. The rotor may also be conical, andtilted so that the movement of the magnets toward and away from theplane of the channel is increased. A conical rotor may also be used inan untilted position, which means that the portion of the channelclosest to the axis of rotation is also closest to the magnets. The tiltangle may be adjustable during use.

The movement of the beads is dictated by the shape of the field as wellas by the motion of the magnets and the geometry of the channel. Thechannel created in a solid substrate may be made using any suitabletechnique, such as milling, molding, extrusion, and the like, andcombinations of techniques. Such channels can be made in plastic, glass,silicon or other materials as long as the magnetic field can passthrough one side of the channel. The channel can also be composed oftubing made of glass, metal, and/or plastic.

The dimensions of the channel can be designed to change the flowvelocity in the different regions of the channel, and consequently tomanipulate the ratio of flow shear to magnetic field strength. Forexample, a channel may have openings at opposing ends and a diameterthat is narrower near the opposing ends than in a center of the channelin order to reduce the flow velocity between the ends of the channel.Reducing the flow velocity can also be used to extend the time that thebeads are in contact with different reagents for sample processing at aconstant flow rate and/or to reduce the sheer forces on the beads. Thebead trap-and-mixer is operable with straight as well as curvedchannels. If retention of a constant angle during the sweep is desired,a horseshoe-shaped channel can be used. Straight channels can haveadvantages for moving beads across the channel or for simplification ofmanufacture or integration into more complex systems.

FIG. 1 illustrates an exemplary embodiment of a magnetic beadtrap-and-mixer. A rotor 1 includes three permanent magnets 2. A topplate 6 and a bottom plate 6 define the sides of a channel 7. The topplate includes an inlet 4 and outlet 5 for the channel 7.

FIG. 2 shows the “catch and release” mixing of magnetic beads. In (a),beads flow through the chamber and become trapped by the magnetic field.In (b), the field created by a first magnet captures the beads, anddrags them upstream as the rotor rotates. During capture, the magnet isrotated so that the magnetic field moves against the direction of flow.In (c), the beads are swept upstream by the magnetic field untilreaching the upstream end or the channel, where the rotation of thefirst magnet moves the field away from the channel. The spinning rotorbrings a second magnet into position at the right side of the drawing.In (d), the beads have been temporarily released and travel with fluidflow through an area of low magnetic field between the magnets. In (e),the beads are captured by the field created by a second magnet, and thecycle can begin again. This operation has been performed with individualmagnets as shown in the figure. It can also be performed using more thanone magnet at each position in order to increase the field strengthsextending into the channel. Magnets can have similar or different fieldstrengths and/or any suitable dimensions

FIG. 3 shows the release of magnetic beads, accomplished by reversingthe direction of rotation of the rotor as compared to FIG. 2. In (a),the magnet begins to move towards the outlet at the downstream end ofthe channel, and the magnetic field concentrates the beads in the streamas they flow toward the downstream end of the channel. In (b), themagnetic field sweeps the beads to the downstream end of the chamber andthe area of high magnetic field begins to be moved away from thechannel. In (c), the beads are released and free to flow out of thechamber for any downstream processing and/or analysis.

Anderson, U.S. patent application Publication No. 2008/0217254,discloses a rotary magnetic bead trap which is connected to a massspectrometry system. Anderson's device requires pairs of magnets withopposing magnetic poles in contact with each other, thereby creating amagnetic field gradient focused on a single point between N/S(north/south) magnet pairs. Because of the point-shaped magnetic field,Anderson's tube or lumen must be positioned in a circular path over therotating magnet carrier so that the magnetic trapping regions arepositioned in the center of the channel. FIG. 4 shows the magneticfields resulting from the arrangement of pairs of magnets 42 and 43embedded in a rotor 41 touching each other at a single point and withtheir magnetic poles in opposite directions. This organization of themagnets focuses the highest strength of the magnetic field to a point44. As a result of this design, the only way to move the beads from sideto side in the channel is to create a serpentine channel deviatingslightly from “the ideal circular profile followed by the magnetic trapregions.” An additional aspect of these concentrated point-shapedtrapping regions is that they collect the magnetic beads into clumpsthat are moved periodically upstream. Since the used beads are sent towaste or collected solely for later use, the resulting aggregation isnot perceived as a problem in Anderson. In contrast, aspects of theapparatus described herein generate a magnetic field extending entirelyacross the diameter of the channel, thus reducing the aggregation ofbeads that is undesirable in many applications. The shape of thechannels in the current invention is not limited by the need toaccommodate a circular arrangement of point-shaped magnetic traps.Anderson also requires a curved tube, whereas the present apparatusoperates effectively with a straight channel, and moreover Andersonfails to appreciate the advantages provided by channels havingparticular contours, such as narrower ends.

FIG. 5 shows the magnetic fields 54 in an embodiment having magnets 52and 53 arranged in a rotor 51 such that a magnetic field 54 is createdthat is long enough to extend across the flow channel. It is notnecessary that the magnets be in contact with one another. The magnetscan be arranged with poles in the same or opposite directions as long asthe magnetic field at areas of high magnetic field extend far enoughinto the channel to capture the magnetic beads under flow conditions andthe areas between the magnets generate sufficiently low magnetic fieldin the channel to allow release of the magnetic beads.

FIG. 6 shows how a linear magnetic field may be used to move the beadsacross a channel as well as longitudinally upstream or downstream, thusenhancing the exposure to the fluid in the channel. The magnetic field64 is shown here with a straight channel 61 and a single bead 65. Theflow is from left to right in the stream and the field is moved fromright to left. Initially, the magnetic field tends to push the beadtoward the side of the channel further from the center of the magnetrotation, but as the rotation continues, the bead is dragged toward theopposite side of the channel.

Example 1

Comparison of capture of fluorescent magnetic beads using differentconfigurations of linear magnetic fields, termed configuration A wherethe poles all point in the same direction (e.g. N/N, N/N, N/N, N/N),configuration B with poles pointed in an alternating configuration (e.g.N/S, S/N, N/S, S/N), and configuration C with opposite pairs of polespaired (e.g. N/S, N/S, N/S, N/S).

In order to visualize the magnetic fields induced by the differentarrangements of the magnets, the linear magnets affixed in the rotatingtrap were removed from under the microfluidic channel and placed under aclear dish containing iron filings and photographed, with FIG. 7Ashowing configuration A, FIG. 7B showing configuration B, and FIG. 7Cshowing configuration C. The photographs suggest that configuration Aproduced a field that extends further into the microchannel to improvethe capture while maintaining regions of low field to permit releasewhen the field is swept in the same direction as the flow. The photo ofconfiguration B suggests that the field required for capture does notextend as far, but that the low field regions necessary for release aremaintained. The photo of configuration C suggests that a microchannelplaced over a region with sufficient field for capture would notexperience a magnetic field sufficiently low for release at any time.

The configurations were tested to effectiveness in trapping andreleasing magnetic beads. Linear magnetic fields were created forsweeping through the fluid passing through a microchannel. The abilityof the fields to capture 6.5 micron fluorescent magnetic beads againstthe direction of flow and retain them was measured, along with thenumber of the beads released when the direction of the magnet rotationwas reversed or when the magnet was removed altogether. Ideally, thebeads would be retained during the capture phase as the magnetic fieldwas swept upstream and released as the magnetic field was sweptdownstream, without the necessity to physically remove the magnets.

Capture takes place when the magnets are positioned in a rotating discimmediately below the microchannel and are rotating in the directionopposite of the flow through the channel. Magnetic release is the stagewhere magnetic beads previously captured by the magnets are released byreversing the direction of magnet rotation. Free release is the flow ofbeads through the microchannel after the magnetic field is removed. FIG.8 shows the results collected: dark gray bars depict data using themagnets positioned all in the same direction (configuration A), lightgray bars indicate data using magnets in pairs with opposite poles(configuration B), and the medium gray bars depict data using magnets inconfiguration C.

The best results were achieved with the “same” configuration, where allthe magnets are oriented with the poles in the same direction (N/N, N/N,N/N, N/N). As is seen in the graph, the concentration of beads/μLexiting the channel was reduced during capture and increaseddramatically during magnet-assisted release. Capture of beads continuedfor ˜20 minutes with a 11 μl/min flow rate.

The second best result was achieved using with the “alternating”configuration, where the adjacent magnets in a pair had opposite poleorientations, and neighboring pairs were minor images of each other(N/S, S/N, N/S, S/N). While the capture was not as efficient as in thefirst configuration, a dramatic release of beads did occur when thedirection of the sweeping magnetic field was reversed. Capture of beadsoccurred for ˜10 minutes at a 11 μl/min flow rate.

In the third configuration, “opposite”, the magnets were arranged sothat every magnet has a pole orientation opposite of its two neighbors(N/S, N/S, N/S, N/S). While the beads were captured, they were notreleased when the rotation of the magnets was reversed. However, therewas a dramatic release of beads when the magnets were pulled completelyout of range of the channel, indicating that the beads were captured,but the magnets did not allow them to escape the channel during theperiod of reversed rotation of the magnets. Capture of beads occurredfor ˜12-15 minutes at a 11 μl/min flow rate.

The apparatus described herein enjoys several advantages over prior artdevices. The simple design and use of permanent magnets permit operationby battery power, for example in a portable device. No significant heatis generated, unlike electromagnetics, so that heat sinks are notrequired and the possibility of degradation of the sample is reduced.The actuation of the trap by use of a reversible motor avoids the needfor specialized armatures and/or plumbing. The design has little or nodead volume, without requiring deep alcoves. Furthermore, the designresults in excellent mixing, in that the repeated “catch and release”cycle allows the beads to spend a period of time free so that their fullsurfaces can be in full contact with the solution. In addition, duringtheir migration upstream, they are being pulled against the solutionflow, increasing the portion of the solution that they come in contactwith compared to beads held in one spot in a channel.

All publications mentioned herein are hereby incorporated by referencefor the purpose of disclosing and describing the particular materialsand methodologies for which the reference was cited.

Although the present invention has been described in connection withpreferred embodiments thereof, it will be appreciated by those skilledin the art that additions, deletions, modifications, and substitutionsnot specifically described may be made without departing from the spiritand scope of the invention. Terminology used herein should not beconstrued as being “means-plus-function” language unless the term“means” is expressly used in association therewith.

1. A magnetic bead trap-and-mixer comprising: a straight channel havingopenings at opposing ends, and a rotor adjacent to the channel andcomprising a permanent magnet, wherein the rotor is adapted to apply amagnetic field to the channel of sufficient strength to direct themovement of magnetic beads therein.
 2. The magnetic bead trap-and-mixerof claim 1, wherein the rotor is configured so that said magnetic fieldthat extends entirely across the channel.
 3. The magnetic beadtrap-and-mixer of claim 1, wherein the rotor is configured so that areasof strong magnetic fields alternate with areas of very weak magneticfields.
 4. The magnetic bead trap-and-mixer of claim 1, wherein therotor comprises at least two permanent magnets.
 5. The magnetic beadtrap-and-mixer of claim 4, wherein the at least two magnets havemagnetic poles all oriented in the same direction with respect to thechannel.
 6. The magnetic bead trap-and-mixer of claim 1, wherein thepermanent magnet is a single magnet that wraps around the channel. 7.The magnetic bead trap-and-mixer of claim 1, wherein the rotor has aplane of rotation that is tilted or adjustable with respect to an axisof the channel.
 8. The magnetic bead trap-and-mixer of claim 1, whereinthe channel has a diameter that is narrower near the opposing ends thanin a center of the channel.
 9. A magnetic bead trap-and-mixercomprising: a channel having openings at opposing ends and a diameterthat is narrower near the opposing ends than in a center of the channel,and a rotor adjacent to the channel and comprising a permanent magnet,wherein the rotor is adapted to apply a magnetic field to the channel ofsufficient strength to direct the movement of magnetic beads therein.10. The magnetic bead trap-and-mixer of claim 9, wherein the rotor isconfigured so that areas of strong magnetic fields alternate with areasof very weak magnetic fields.
 11. The magnetic bead trap-and-mixer ofclaim 9, wherein the rotor is configured so that said magnetic fieldthat extends entirely across the channel.
 12. The magnetic beadtrap-and-mixer of claim 9, wherein the rotor comprises at least twopermanent magnets.
 13. The magnetic bead trap-and-mixer of claim 12,wherein the at least two magnets have magnetic poles all oriented in thesame direction with respect to the channel.
 14. The magnetic beadtrap-and-mixer of claim 9, wherein the permanent magnet is a singlemagnet that wraps around the channel.
 15. The magnetic beadtrap-and-mixer of claim 9, wherein the rotor has a plane of rotationthat is tilted or adjustable with respect to an axis or plane of thechannel.
 16. The magnetic bead trap-and-mixer of claim 9, wherein thechannel is straight.
 17. A magnetic bead trap-and-mixer comprising: achannel having openings at opposing ends, and a rotor adjacent to thechannel and comprising a permanent magnet, wherein the rotor is adaptedto apply a magnetic field to the channel of sufficient strength todirect the movement of magnetic beads therein, and the rotor generatesin the channel areas of areas of strong magnetic fields alternating withareas of very weak magnetic fields and the strong magnetic fields extendentirely across the channel.
 18. The magnetic bead trap-and-mixer ofclaim 17, wherein the rotor has a plane of rotation that is tilted oradjustable with respect to an axis or plane of the channel.
 19. Themagnetic bead trap-and-mixer of claim 17, wherein the channel isstraight.
 20. The magnetic bead trap-and-mixer of claim 17, wherein thechannel has a diameter that is narrower near the opposing ends than in acenter of the channel.